In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Alternative Splicing↗

PubMed1998

Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination.

The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.

Base Sequence↗

PubMed1998

Enhanced and specific gene expression via tissue-specific production of Cre recombinase using adenovirus vector.

A tissue-specific promoter is potentially valuable for the study of specific gene function and for gene therapy, as it permits a linked cytotoxic or any other gene to be expressed specifically in target cells. The expression levels of such promoters are generally low, and we have therefore developed a novel and general method to enhance the expression level of a tissue-specific promoter while maintaining specificity. We constructed a "regulator" recombinant adenovirus (rAd) producing the site-specific recombinase Cre under the control of the hepatocarcinoma-specific alpha-fetoprotein (AFP) promoter. The rAd was infected to AFP-producing cells together with a "target" rAd containing a Cre-activating potent expression unit. In in vitro experiments, the double infection method gave about 50-fold higher expression than the single rAd infection directly driven by the AFP promoter, while maintaining strict specificity to AFP-producing cells. The enhanced and specific expression was also observed in in vivo tumor models. This method may contribute not only to the establishment of specific gene therapies but also to basic study for elucidating cell-type specific gene functions.

Adenoviridae↗

PubMed1998

Selective inhibition of the chymotrypsin-like activity of the 20S proteasome by 5-methoxy-1-indanone dipeptide benzamides.

Potent inhibitors of the 20S proteasome that contain a novel indanone head group coupled to di and tripeptides are described. These compounds are the first proteasome inhibitors have demonstrated high selectivity for the chymotrypsin-like activity of the 20S proteasome.

Benzamides↗

PubMed1998

W474C amino acid substitution affects early processing of the alpha-subunit of beta-hexosaminidase A and is associated with subacute G(M2) gangliosidosis.

Mutations in the HEXA gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), that abolish Hex A enzyme activity cause Tay-Sachs disease (TSD), the fatal infantile form of G(M2) gangliosidosis, Type 1. Less severe, subacute (juvenile-onset) and chronic (adult-onset) variants are characterized by a broad spectrum of clinical manifestations and are associated with residual levels of Hex A enzyme activity. We identified a 1422 G-->C (amino acid W474C) substitution in the first position of exon 13 of HEXA of a non-Jewish proband who manifested a subacute variant of G(M2) gangliosidosis. On the second maternally inherited allele, we identified the common infantile disease-causing 4-bp insertion, +TATC 1278, in exon 11. Pulse-chase analysis using proband fibroblasts revealed that the W474C-containing alpha-subunit precursor was normally synthesized, but not phosphorylated or secreted, and the mature lysosomal alpha-subunit was not detected. When the W474C-containing alpha-subunit was transiently co-expressed with the beta-subunit to produce Hex A (alphabeta) in COS-7 cells, the mature alpha-subunit was present, but its level was much lower than that from normal alpha-subunit transfections, although higher than in those cells transfected with an alpha-subunit associated with infantile TSD. Furthermore, the precursor level of the W474C alpha-subunit was found to accumulate in comparison to the normal alpha-subunit precursor levels. We conclude that the 1422 G-->C mutation is the cause of Hex A enzyme deficiency in the proband. The resulting W474C substitution clearly interferes with alpha-subunit processing, but because the base substitution falls at the first position of exon 13, aberrant splicing may also contribute to Hex A deficiency in this proband.

Adolescent↗

PubMed1998

The relaxation effects of ginseng saponin in rabbit corporal smooth muscle: is it a nitric oxide donor?

OBJECTIVE: To determine whether crude extracts of ginseng saponin (GCS), containing the active ingredients from Panax ginseng and used as an aphrodisiac in oriental countries, relax corpus cavernosal smooth muscle in the rabbit. MATERIALS AND METHODS: Corpus cavernosal strips were prepared from rabbit penises. Isometric tension changes, recorded with a pressure transducer, in response to various drugs and electrical stimulation were assessed in an organ chamber, after active muscle tone had been induced by 10 micromol/L phenylephrine. RESULTS: GCS (0.2-8.0 mg) relaxed the smooth muscle of rabbit corpus cavernosum (SMRCC) pre-contracted with phenylephrine in a dose-dependent manner. GCS at 0.75 mg significantly enhanced the relaxation of SMRCC induced by electrical field stimulation. The relaxation induced by 0.2-8.0 mg GCS was significantly attenuated by atropine (1 micromol/L), methylene blue (100 micromol/L) and N-omega-nitro-L-arginine methyl ester (L-NAME, 10 micromol/L). However, there was no significant difference in the attenuation of GCS-induced relaxation of SMRCC by adding vasoactive intestinal peptide antagonists or indomethacin. In addition, the decreasing rate of GCS-induced relaxation of SMRCC by methylene blue and L-NAME was greater than that by atropine. L-arginine (10 mmol/L) reversed the inhibitory effect induced by L-NAME (1 mmol/L) on the attenuation of GCS-induced relaxation. CONCLUSIONS: These data suggest that GCS, as a nitric oxide donor, induces the relaxation of SMRCC through the L-arginine/nitric oxide pathway. For the clinical application of ginseng saponin, further studies are required to clarify the active subfraction(s) of GCS.

Animals↗

PubMed1998

An unusual case of renal cell carcinoma with two rare metastases.

It is well known that renal cell carcinoma metastasizes to unusual sites. We report a case in which two rare sites of metastasis, the maxilla and contralateral ureter, appeared in a single patient.

Aged↗

PubMed1998

AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans.

BACKGROUND: Animal studies suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-kainate (AMPA-KA) receptors are involved in pain processing. The effects of the competitive AMPA-KA antagonist LY293558 in two types of experimental pain in human volunteers, brief pain sensations in normal skin, and mechanical allodynia-pinprick hyperalgesia were studied after the injection of intradermal capsaicin. METHODS: Brief intravenous infusions of the competitive AMPA-KA antagonist LY293558 were given to 25 healthy volunteers to examine acute toxicity and analgesic effects. Fifteen volunteers then entered a double-blinded, three-period crossover study. In a Phase II study, LY293558 infusions (100% maximally tolerated dose vs. 33% maximally tolerated dose vs. placebo) began 10 min after intradermal injection of 250 microg capsaicin in volar forearm. Spontaneous pain, areas of mechanical allodynia and pinprick hyperalgesia, and side effects were determined every 5 min for 60 min. RESULTS: The median maximally tolerated dose was 1.3 +/- 0.4 (range, 0.9-2.0) mg/kg. Tests of cognitive and neurological function were unchanged. Dose-limiting side effects were hazy vision in 95% of volunteers and sedation in 40%. There were no significant changes in electrical or warm-cool detection and pain thresholds or heat pain thresholds. LY293558 had little effect on brief pain sensations in normal skin. Both high and low doses of LY293558 significantly reduced pain intensity, pain unpleasantness, and the area in which light brush evoked pain after intradermal capsaicin. There was a trend toward a dose-response effect of LY293558 on the area in which pinprick evoked pain after intradermal capsaicin, which did not reach statistical significance. CONCLUSIONS: The authors infer that AMPA-KA receptor blockade reduces the spinal neuron sensitization that mediates capsaicin-evoked pain and allodynia. The low incidence of side effects at effective doses of LY293558 suggests that this class of drugs may prove to be useful in clinical pain states.

Adult↗

PubMed1998

Accidental digital injection of adrenaline from an autoinjector device.

Explore the source record for details and available documents.

Adrenergic alpha-Antagonists↗

PubMed1998

Development and maintenance of the gut-associated lymphoid tissue (GALT): the roles of enteric bacteria and viruses.

GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from "virgin" B cells in neonates. Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to "activation" of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and "constitutively" cytotoxic T cells.

Animals↗

PubMed1998

Tau interacts with src-family non-receptor tyrosine kinases.

Tau and other microtubule-associated proteins promote the assembly and stabilization of neuronal microtubules. While each microtubule-associated protein has distinct properties, their in vivo roles remain largely unknown. Tau is important in neurite outgrowth and axonal development. Recently, we showed that the amino-terminal region of tau, which is not involved in microtubule interactions, is important in NGF induced neurite outgrowth in PC12 cells. Here we report that a proline rich sequence in the amino terminus of tau interacts with the SH3 domains of fyn and src non-receptor tyrosine kinases. Tau and fyn were co-immunoprecipitated from human neuroblastoma cells and co-localization of tau and fyn was visualized in co-transfected NIH3T3 cells. Co-transfection of tau and fyn also resulted in an alteration in NIH3T3 cell morphology, consistent with an in vivo interaction. Fyn-dependent tyrosine phosphorylation of tau occurred in transfected cells and tyrosine phosphorylated tau was identified in human neuroblastoma cells as well. Our data suggest that tau is involved in signal transduction pathways. An interaction between tau and fyn may serve as a mechanism by which extracellular signals influence the spatial distribution of microtubules. The tyrosine phosphorylation of tau by fyn may also have a role in neuropathogenesis, as fyn is upregulated in Alzheimer's disease.

3T3 Cells↗

PubMed1998

Emphysematous cystitis.

Emphysematous cystitis is characterized by gas collection within the bladder wall and lumen. We report two cases of emphysematous cystitis of the urinary bladder in a 67-year-old and a 63-year-old women. They presented with bladder irritation symptoms such as dysuria, hematuria and frequency. Urinalysis showed pyuria. Cystoscopic examination revealed that bladder mucosa was studded with vesicles varying in size and arranged in clumps. CT scans of the pelvis showed mottled gas bubbles within the bladder. They were treated with antibiotics. Four days after the treatment, the symptoms subsided and plain abdominal film showed no evidence of gas shadows in the pelvic cavity.

Aged↗

PubMed1998

Conversion of normal human oral keratinocytes to tumorigenic cells is associated with the acquisition of resistance to TGF-beta.

Normal human epithelial cells cannot proliferate and undergo apoptosis in the presence of transforming growth factor-beta (TGF-beta) in vitro, but many human epidermoid cancer cells are resistant to TGF-beta. Resistance to TGF-beta may thus, in part, be responsible for uncontrolled proliferation of cancer cells. Though detailed mechanisms for the resistance of cancer cells to TGF-beta remain unknown, resistance may be due to decreased expression of TGF-beta receptors from cancer cells. To investigate this possibility, we determined the expression of TGF-beta and type II TGF-beta receptor in primary normal human oral keratinocytes (NHOK), human papillomavirus-immortalized human oral keratinocytes (HOK-16B) and two tumor cell lines derived from HOK-16B (CTHOK-16B-BaP and CTHOK-16B-DMBA). Our results show that (1) the cellular and secretory TGF-beta levels in immortalized and tumor cells were notably lower than in NHOK and (2) the level of type II TGF-beta receptor of the tested cells was similar to each other. Taken together, the conversion of NHOK to tumorigenic cells may, in part, be due to the acquisition of NHOK resistance to TGF-beta through underexpression of this cytokine.

Apoptosis↗